The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood-Brain Barrier in Glioblastoma

Abstract

Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide.

Experimental design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging.

Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors.

Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM.

Figure 1. Initial in vitro evaluation of MK-1775 in a panel of primary GBM lines

A) Western blot analysis of flank tumor GBM xenografts. Pooled lysate from 3 biological replicates of a tumor line were run in each lane for the indicated xenograft models. B) Effects of graded concentrations of MK-1775 were assessed in a CyQuant assay for 5 xenograft lines. Results shown are the mean ± SEM from three independent experiments. Statistically significant differences in fluorescence following a given treatment relative to control for each line are shown with * denoting p-value < 0.05, C) The sensitivity of GBM6, GBM12 and GBM22 to MK-1775 was assessed in a primary neurosphere assay. The number of neurospheres following drug treatment in 3 independent experiments is shown as the mean ± SEM. Statistically significant differences are noted comparing GBM22 vs. GBM12 and GBM22 vs. GBM6 at 100 and 300 nM. D) The impact of a 24 hour pre-treatment of MK-1775 on p-Cdk1 was assessed by western blotting. Because of low basal phosphorylation, GBM22 was irradiated (RT) with 10 Gy.

Figure 2. DNA damage analysis in GBM6 and GBM22 after MK-1775 treatment

A) γH2AX foci formation in GBM6 and GBM22 were assessed 24 h after a single treatment of 300 nM MK-1775. B) All cells with γH2AX staining (>20 foci/nuclei or pan-nuclear) or just those only with pan-nuclear γH2AX staining were quantitated and shown as the mean ± SEM from 3 independent experiments, magnification bar is 25 µm and * indicates p<0.05 compared to controls.

Figure 3. In vitro combination of MK-1775 and TMZ in GBM6 and GBM22

A) GBM6 and B) GBM22 cells were treated simultaneously with graded concentrations of TMZ and MK-1775 and then analyzed in a CyQuant assay. C) Western blot evaluation of GBM6 and GBM22 short-term explant cultures 24 hours after treatment with either MK-1775 or TMZ or the combination. Results are representative of 3 independent experiments, * indicates p<0.05 for a given concentration of MK-1775 relative to TMZ/control alone.

Figure 4. Efficacy of MK-1775 in combination with RT and TMZ in brain tumors

Mice with established orthotopic GBM22 tumors treated with radiation (RT), TMZ and/or MK-1775 in a single experiment and survivals are presented in 3 graphs. A) The combination of MK-1775 (50 mpk twice daily Days 1–5 and 8–12) alone or concurrent with RT (2 Gy/day, Days 1–5 and 8–12). B) The same dosing regimen with TMZ (20 mpk/day given Days 1–5 and 8–12). C) Treatment with TMZ alone (50 mpk daily Days 1–5, 29–33 and 57–61) with MK-1775 (50 mpk twice daily Days 1–5, 29–33 and 57–61).

Figure 5. Evaluation of MK-1775 distribution in brain and tumor tissues

A) Mice received a single oral dose of MK-1775 (50 mg/kg) and blood and normal brain were collected at the indicated time-points. Results are the mean ± SD at each time-point (n=3 mice per point). Mice with established GBM22 B) orthotopic or C) flank tumors were euthanized two hours after a single dose of MK-1775 (200 mg/kg) and processed for MALDI-MSI. Red and green color intensities indicate relative levels of heme (m/z 616.2 ± 0.2) and MK-1775 (m/z 501.2 ± 0.2), respectively. Black dotted lines delineate tumor tissue in H&E stained sister sections. Results are representative of 3 mice in each condition.

Figure 6. MK-1775 and TMZ efficacy evaluation in GBM22 flank xenografts

A) Mice with established GBM22 flank xenografts were treated with TMZ 50 mpk with or without MK-1775 50 mpk twice daily on Days 1–5, 29–31 and 57–61. B) In the same experiment, extended dosing of MK-1775, given 5 days a week from Day 1 until Day 110, was evaluated alone and in combination with the same TMZ dosing regimen used in A. C) Animals with established GBM22 flank xenografts were treated for days 1–5 with TMZ 50 mg/kg/day, with or without MK-1775 dosed at 50 mg/kg twice daily for days 1–5 then days 8–12, and harvested on the days listed. Equal amounts of protein from 3 biological replicates were pooled and run in an individual lane as indicated.